Phenotypic plasticity and genetic diversity shed light on endemism of rare Boechera perstellata and its potential vulnerability to climate warming

Abstract The rapid pace of contemporary environmental change puts many species at risk, especially rare species constrained by limited capacity to adapt or migrate due to low genetic diversity and/or fitness. But the ability to acclimate can provide another way to persist through change. We compared the capacity of rare Boechera perstellata (Braun's rockcress) and widespread B. laevigata to acclimate to change. We investigated the phenotypic plasticity of growth, biomass allocation, and leaf morphology of individuals of B. perstellata and B. laevigata propagated from seed collected from several populations throughout their ranges in a growth chamber experiment to assess their capacity to acclimate. Concurrently, we assessed the genetic diversity of sampled populations using 17 microsatellite loci to assess evolutionary potential. Plasticity was limited in both rare B. perstellata and widespread B. laevigata, but differences in the plasticity of root traits between species suggest that B. perstellata may have less capacity to acclimate to change. In contrast to its widespread congener, B. perstellata exhibited no plasticity in response to temperature and weaker plastic responses to water availability. As expected, B. perstellata also had lower levels of observed heterozygosity than B. laevigata at the species level, but population‐level trends in diversity measures were inconsistent due to high heterogeneity among B. laevigata populations. Overall, the ability of phenotypic plasticity to broadly explain the rarity of B. perstellata versus commonness of B. laevigata is limited. However, some contextual aspects of our plasticity findings compared with its relatively low genetic variability may shed light on the narrow range and habitat associations of B. perstellata and suggest its vulnerability to climate warming due to acclimatory and evolutionary constraints.


| INTRODUC TI ON
Biodiversity is at risk worldwide due to dramatic rates of environmental change, often as a result of anthropogenic activities (Knapp et al., 2021;Malhi et al., 2020).Rare species are especially susceptible to extinction in the face of such change (Mouillot et al., 2013), and as such, rare species often drive declines in the biodiversity of communities and systems (Dee et al., 2019).Species can persist through environmental change via migration to more suitable habitat (Chen et al., 2011;Crickenberger & Wetheym, 2018;Hickling et al., 2006;Parmesan, 2006), adaptation to changed conditions in existing habitat (Hamann et al., 2020;Jump & Peñuelas, 2005;Sheth et al., 2018), and/or acclimation to changed conditions (Chevin et al., 2010;Nicotra et al., 2010;Seebacher et al., 2015).
But relative to more common species, the ability of rare species to migrate and/or adapt could be impeded by their low fitness (Boyd, Anderson, et al., 2022;Iverson et al., 2004) and/or genetic diversity (Cole, 2003;Gitzendanner & Soltis, 2000;Leimu & Fischer, 2008).
Given these constraints, the ability to acclimate to relatively rapid environmental change through phenotypic plasticity could be an important pathway to persistence for rare species.
Relative to other taxonomic groups, plants are generally characterized by high levels of plasticity (Sultan, 2000).But plant species, populations, and individuals can exhibit dramatic differences in plasticity that may influence their responses to environmental change in different ways (Balaguer et al., 2001;Cleavitt, 2002;Dangremond et al., 2015;Godoy et al., 2012;Nicotra & Davidson, 2010;Osunkoya & Swanborough, 2001;Pohlman et al., 2005;Stamp & Hatfield, 2020;Sultan, 2000;Valladares et al., 2000Valladares et al., , 2007)).Plasticity has been underexplored in the context of the causes and consequences of plant species rarity (Boyd, Anderson, et al., 2022; but see Boyd, Odell, et al., 2022;Hirst et al., 2017;Liao et al., 2006;Lovell & McKay, 2015;Rutherford et al., 2017), although it has been long suggested that rare species could be constrained by low plasticity relative to more common species (Murray et al., 2002).Such constraints could explain both the narrow ranges and habitat specificity of many rare species, as well as their declines in the face of environmental change (Nicotra et al., 2010).Studies that compare the plasticity of rare and common species in the context of environmental change could test the hypothesis that geographically restricted species and/or habitat specialists may be limited by an inability to acclimate to broader environmental conditions (Boyd, Anderson, et al., 2022) and provide guidance for rare species conservation during a time of rapid environmental change (Bevill & Louda, 1999).Congeneric comparisons could be especially useful in advancing our understanding of the causes and consequences of species rarity by controlling for differences in life history and phylogeny (Combs et al., 2013;Farnsworth, 2006;Godt & Hamrick, 2001;Kunin & Gaston, 1997;Murray et al., 2002).Boechera perstellata (E.L. Braun) Al-Shehbaz (Braun's rockcress) is a rare endemic plant species with a disjunct distribution consisting of a small number of populations along wooded limestone outcrops in central Tennessee and north-central Kentucky, USA (USFWS, 1995(USFWS, , 1997(USFWS, , 2004) ) separated by a ~250-km gap (Figure 1a).
Previous research revealed that B. perstellata has very low levels of genetic diversity (Baskauf et al., 2014) but that study did not include relative comparisons with more common species.In contrast, widespread B. laevigata (Muhl.Ex Willd.)Al-Shehbaz (smooth rockcress) occurs throughout much of the eastern United States (Figure 1b) as well as southern Quebec in habitat that is largely similar to that of B. perstellata (Al-Shehbaz & Windham, 2010;Bloom et al., 2002) but across a broader range of substrates (Kiefer et al., 2009).Populations of B. laevigata also have been reported along railroad tracks suggesting that its distribution in some locations may have been influenced by human activities (Kiefer et al., 2009).
To investigate the role of acclimation as a cause and/or consequence of species rarity, we compared the phenotypic plasticity of rare B. perstellata and widespread B. laevigata in response to a suite of abiotic factors-light, temperature, and water availability.We also compared the evolutionary potential of these species through investigations of their population-level genetic diversity.Given its narrow geographic distribution and less diverse habitat associations than B. laevigata, we hypothesized that rare B. perstellata would have lower plasticity and genetic diversity than its more common congener.
In particular, we expected B. perstellata to have comparably lower plasticity in biomass allocation traits associated with light and water acquisition in the context of the availability of these resources.In response to warming, we expected B. perstellata to exhibit lower plasticity in traits associated with size and leaf morphology, which have been found to be particularly responsive to temperature (see Stotz et al., 2021).We also expected that populations of B. perstellata would exhibit less intraspecific variation in plasticity and withinpopulation genetic diversity-as causal and/or consequential to rarity-than would populations of B. laevigata, although we expected some significant divergence between the disjunct Tennessee and Kentucky populations of B. perstellata.

| Study system
Boechera perstellata is a rare species restricted to just three counties each in the Central Basin region of Tennessee, USA and the Bluegrass region of north-central Kentucky, USA (USFWS, 2004(USFWS, , 2018)).This rare species is associated with almost dry to moderately moist, shady, steep slopes on limestone outcrops, often in sheltered areas with limited competition from surrounding vegetation (USFWS, 2004(USFWS, , 2018)).At present, there are 47 occurrences of B. perstellata known from throughout its range with the vast majority occurring in Kentucky (USFWS, 2018).While most occurrences consist of few individuals, some are comprised of >1000 individuals (NatureServe, 2005).Given its small geographic range and narrow habitat specificity (i.e., limestone outcrops) but large size of some populations, we categorize B. perstellata as "endemic" (see Rabinowitz, 1981), which is the most common type of species rarity (May, 1988;Rabinowitz, 1981).Given its endemism and associated conservation concerns, B. perstellata is listed as federally endangered (USFWS, 2018).Like its rare congener, widespread B. laevigata often occurs on limestone rock outcrops, but this species also can be found growing on open rocky or gravel areas throughout a wider range that encompasses much of eastern North America (Al-Shehbaz, 1988).
Despite their phylogenetic relatedness, comparisons of Boechera species can be complicated by differences in their life histories and reproductive and ploidy patterns.While the genetic diversity of B.
perstellata has been compared with that of short-lived perennials in particular (Baskauf et al., 2014), its lifespan is not well known beyond the persistence of woody old growth for several years (Braun, 1956).
Depending on plant size and resource availability, B. laevigata has been described as being able to function as a short-lived perennial or biennial (Bloom et al., 2002(Bloom et al., , 2003)).While most Boechera species are sexual diploids, some species are polyploid, and some reproduce asexually through seed (apomixis; Dobeš et al., 2006).Both B. perstellata and B. laevigata are typically diploid, sexually reproducing species, and B. laevigata is known to be capable of reproducing by both outcrossing and selfing (Bloom, 1988).Recent evidence also suggests that a few populations of this species are reproducing by apomixis (Carman et al., 2019;M. Windham, personal communication).The model organism Arabidopsis thaliana is in the same family (Brassicaceae) as Boechera species, and the Boechera genus has become a model system for testing ecological and evolutionary questions given its relatively small genomes and occurrence in natural habitats throughout North America (Rushworth et al., 2011;Schranz et al., 2007).S2).

| Environmental treatments
Following transplantation, we programmed the four growth chambers to allow us to assess plasticity of B. perstellata and B. laevigata in response to light, temperature, and water as modified from methods previously described by Boyd, Odell, et al. (2022).We generally aimed to impose contrasting levels of these abiotic factors across which plasticity could be tested that were also informed by the general direction and specifics of likely environmental change when possible, although this determination was more apparent for some factors than others.We programmed an "ambient" chamber to be used in all of the plasticity assessments to provide temperature of 20-30°C (nighttime-daytime) based on regional weather records and a 12-h photoperiod with a maximum daily light level of 250 μmol photons/m 2 /s in accordance with our field measurements of photosynthetically active radiation (PAR) in B. perstellata habitat during the growing season.The availability of water in B. perstellata habitat is a complex abiotic factor to assess due to the potential for smallscale temporal variation associated with precipitation events.But given the generally well-drained substrate associated with B. perstellata, we imposed a level of water stress as a baseline in our plasticity experiments by watering pots in the ambient chamber to 50% field capacity every 2 days as determined by weighing a subset of pots of each species in accordance with previously described methods (see Boyd, Odell, et al., 2022).The other three growth chambers were programmed to provide the same conditions as the "ambient" chamber but each with a contrasting level of a single condition (light, temperature, or water) across which plasticity could be assessed.Specifically, in one chamber, we doubled light availability to a maximum daily level of 500 photons μmol/m 2 /s to represent a dramatic increase in light, as could be associated with timber harvesting and/or other land-use change associated with deforestation.
In another chamber, we simulated average projections of global temperature increase for this century (Pörtner et al., 2022) by exposing plants to 22-32°C (nighttime-daytime).In the final chamber, we doubled water availability by watering plants to 100% field capacity every 2 days.Projections suggest that precipitation amounts and heavy precipitation events will increase in eastern North America (IPCC, 2023); however, we concede that the effects of such change on water availability will be complicated by edaphic factors and effects on evapotranspiration.As such, while allowing us to test for plasticity, the "real world" translation of our water treatment levels is limited.We grew all individuals in the growth chambers for 4 months following treatment initiation during which time growth data were collected.To minimize any effects of chamber and pseudoreplication (Gibson, 2014), we reassigned treatment levels to each chamber monthly and moved all plants accordingly.Within chambers, we rotated the positions of pots each week to control for spatial differences in environmental conditions.

| Growth, allocation, and leaf morphology measures
We recorded plant height and counted the numbers of leaves of each individual at 4 months after treatment initiation.We then harvested all individuals to yield measures of growth and biomass allocation.We quantified root length (cm) as the distance from the tip of the longest root to the beginning of the green shoot when freshly harvested plants were held upright.The single youngest fully expanded leaf from each individual was removed, scanned to determine its area, and dried in a laboratory oven to calculate specific leaf area (SLA; cm 2 /g) for each individual.Concurrently, we separated the remains of each harvested plant into shoots and roots, which were also dried to determine shoot dry mass (g), root dry mass (g), and total dry mass (g).We included the dry mass of leaf material removed for SLA measurements and genetic investigations (described in later subsections) in our determinations of shoot dry mass.We calculate mass-based root-to-shoot ratio (RSR mass ; g/g) by dividing root dry mass by shoot dry mass, length-based root-to-shoot ratio (RSR length ; cm/cm 2 ) by dividing root length by shoot length, and specific root length (SRL; cm/g) by dividing root length by root dry mass.

| Analyses of survival and trait plasticity
We analyzed the probability of survival to harvest as a function of species, growth chamber treatment, and their interaction in a generalized linear mixed model framework with a binomial distribution and a random effect for source population in the glmer function of the lme4 R package.
To analyze plasticity of growth, allocation, and leaf morphological traits, we fit linear mixed-effects models using the lmer function of the R package lme4 (Bates et al., 2015) with species, abiotic treatment (i.e., light, temperature, and water), and their interaction as explanatory factors.We included population as a random effect in these models.A significant interaction indicated that B. perstellata and B. laevigata responded differently to a change in the environmental factor (i.e., that the two species exhibited differences in plasticity; see Boyd, Odell, et al., 2022).To account for the numerous traits analyzed to assess plasticity, we used corrected p-values to minimize the false discovery rate (FDR; Benjamini & Hochberg, 1995).Results of statistical tests were considered significant if FDR-corrected p ≤ .05.When there was a significant species × abiotic treatment interaction, we contrasted estimated means of each species between abiotic treatment levels with the R package emmeans (ver.1.8.7).
We used a relative distance plasticity index (RDPI; see Valladares et al., 2006) to calculate plasticity of traits in the context of our light, temperature, and water treatments as previously detailed in these contexts by Boyd, Odell, et al. (2022).The RDPI, which can range from 0 (no plasticity) to 1 (maximum plasticity), is based on the absolute phenotypic distances of 1.8.7 genotypes across different environments and allows for statistical comparison of plasticity for species and populations (Valladares et al., 2006).To assess the potential influence of plasticity on fitness, we conducted acrossenvironment multivariate genotypic selection analysis (Stinchcombe et al., 2004;Van Kleunen & Fischer, 2001) with the lmer (linear mixed model) function of the R package lme4 (ver.1.1-21; Bates et al., 2015).We focused these analyses on traits with significant effects of abiotic factor or abiotic factor × species interactions (i.e., traits for which we found significant evidence for plasticity).We analyzed fitness as a function of mean trait values, RDPI, species, and two-way interactions between mean trait values and species and RDPI and species in separate models for each manipulated environmental condition (i.e., temperature and water).The average total dry biomass calculated across ambient and manipulated environmental conditions (e.g., ambient and increased temperature) for each maternal line (i.e., mean total biomass) served as the fitness proxy in these regressions because total biomass was measurable for all individuals included in our experiment and is generally associated positively with reproductive output (Weiner et al., 2009).A significant effect of RDPI in a trait on fitness suggests selection for plasticity in both species if the slope is positive and selection against plasticity if the slope is negative.A significant interaction between species and RDPI suggests that the magnitude or direction of selection differs between species.Because we conducted two separate analyses of selection on plasticity, we used a Bonferroni-corrected α = 0.025 (= 0.05/2) to assess significance and control for type I errors.We used the predictorEffects function of the R package effects (ver. 4.2-0; Fox & Weisberg, 2018) to visualize selection landscapes from these multiple regression models as partial residual plots.

| DNA extraction and microsatellite genotyping
Leaf tissue was collected from all B. perstellata and B. laevigata individuals included in the growth chamber experiments prior to harvest.Collected leaf tissue was dried on silica gel and ground in a bead mill (MM301; Retsch).DNA was extracted from leaf material with the E.Z.N.A. SP Plant DNA Kit (Omega Bio-Tek, Inc.).Previously designed microsatellite primers were initially evaluated using touchdown polymerase chain reaction (PCR) analysis (see Baskauf et al., 2014), except for ICE4, F03, and H06, for which constant annealing temperatures of 50, 50, and 55°C, respectively, were used.
Fluorescent labeling protocols followed Schuelke (2000) as modified by Baskauf et al. (2014), except that six primers (F03, G03, G06, G08, G09, and H06) had a short tag (GTTTCTT) attached to the 5′ end of the reverse primer.PCR products were multiplexed, and fragment analysis was completed with an ABI 3130XL DNA Analyzer (Applied Biosystems) by the University of Tennessee Health Science Center (Memphis, Tennessee, USA).Individuals were genotyped using Gen-eMarker v1.97 (SoftGenetics, LLC).Alleles were manually identified and verified by two people.

| Analysis of genetic diversity
In total, 185 B. perstellata and 151 B. laevigata individuals were included in most analyses.We used GenAlEx v6.503 (Peakall & Smouse, 2006, 2012) to calculate standard genetic diversity statistics, including percentage of polymorphic loci (P), alleles per locus (A), observed (H o ), and Nei's (1978) (Alhusain & Hafez, 2018).We used the randomization-based testing of FSTAT ver.2.9.4 (Goudet, 1995(Goudet, , 2003) ) S2), but the probability of survival varied as a function of treatment.Specifically, individuals across both species had a significantly lower probability of survival with increased watering than in ambient conditions (Figure S1).In contrast, the probability of survival did not differ between species across treatments.The interaction of species and treatment also did not significantly affect survival.No individuals of either species flowered during the experiment.

| Species growth, allocation, and leaf morphology
There were significant differences in several traits of the rare and widespread Boechera species across levels of abiotic treatments (Table S3).Specifically, in all three comparisons (i.e., ambient vs. increased light, ambient vs. increased temperature, ambient vs. increased water), B. perstellata individuals had more leaves and greater SLA than B. laevigata individuals (Table S4).In the ambient versus increased water comparison only, B. laevigata had greater root mass and RSR mass than B. perstellata individuals across treatment levels (Table S4).

| Phenotypic plasticity
We found no evidence for phenotypic plasticity of either B. perstellata or B. laevigata in response to light availability (see results for main effects of abiotic factor and species × abiotic factor interaction in Table S3).However, there were some differences in the plasticity of root growth and allocation of rare and widespread Boechera species in response to both warming and increased water availability (see results for species × abiotic factor interaction in Table S3).In response to warming, widespread B. laevigata allocated less of its total biomass to roots (i.e., lower RMR mass ) but had greater SRL (Figure 2).In contrast, B. perstellata did not exhibit plasticity of these or any other any measured traits in the context of temperature change (Figure 2).In response to increased water availability, individuals of both B. perstellata and B. laevigata produced less root biomass and allocated less of their total biomass to roots (Figure 3).In addition, increased water availability was associated with greater SLA of rare B. perstellata but no significant change in the SLA of B. laevigata (Figure 3).Selection via biomass did not operate significantly on trait plasticity under temperature manipulation (Table S5), but selection did operate significantly on trait plasticity of RSR mass under water manipulation (Table S6).Specifically, there was selection for plasticity of RSR mass (i.e., plasticity of this trait was adaptive) in both Boechera species (Figure S2).

| Genetic diversity
A total of 17 microsatellite loci amplified in all populations for both species and were polymorphic in at least one of the species (Table S7 S3).
Specifically, all but one of the B. perstellata populations clustered together with relatively minor genetic differences, while the three B.
laevigata populations were more widely spaced in the analysis output.Each population was its own genetic cluster, except that the relatively high diversity TN population of B. laevigata contained three genetic clusters and TN1 population of B. perstellata contained two clusters.

| DISCUSS ION
Phenotypic plasticity can influence organismal fitness and species performance by facilitating expansion across diverse abiotic environments and/or persistence in locations experiencing environmental change (Godoy et al., 2012;Nicotra & Davidson, 2010).For rare endemic species, limited phenotypic plasticity could constrain their geographic range and/or habitat specificity, while widespread species with broader habitat breadths could be facilitated by relatively high phenotypic plasticity (Murray et al., 2002).However, our findings suggest that the ability of phenotypic plasticity to elucidate the relative rarity and commonness of B. perstellata and B. laevigata, respectively, is limited.Specifically, we found no evidence of plasticity of any measured traits for either species within the context of light and limited differences in plasticity between species within the context of temperature and water.But we suggest that the differences that we did detect in the plasticity of root traits of rare B. perstellata and widespread B. laevigata in response to those abiotic factors could help to explain differences in their geographic range sizes and habitat associations.
Previous research suggested that temperature plasticity in particular could be especially influential to range size of Boechera species (Lovell & McKay, 2015), although other studies of plasticity of rare and common species within the context of temperature did not reveal similar trends (Hirst et al., 2017).Our significant plasticity findings suggest that widespread B. laevigata may be able to alter its root allocation in response to warming in ways that B.
perstellata cannot.The specific responses of B. laevigata to warming revealed by our study could indicate increased investment in longer but thinner roots, which could increase the acquisition of belowground resources when they are limited with less total root biomass investment (see Ostonen et al., 2007).Although we did not measure soil moisture of pots in our high-temperature treatment level, it is plausible that warming would be associated with availability, but in a direction that contrasts other reported responses of SLA to wetter conditions (see Dwyer et al., 2014;Rosas et al., 2019).
In the face of environmental change, limited plasticity could impede species persistence, especially when migration and/or adaptation are also impeded by low fitness and genetic diversity as is characteristic of many rare species (Boyd, Anderson, et al., In contrast, phenotypic plasticity could provide a pathway to persistence through change, especially relatively rapid environmental change that may outpace the ability of species to adapt to new conditions (Chevin et al., 2010;Snell-Rood et al., 2018).Habitat loss due to disturbances such as development and timber harvesting remains an ongoing threat to rare B. perstellata given its small number of extant occurrences (USFWS, 2004).A lack of phenotypic plasticity of B. perstellata in the context of light (which is shared by widespread B. laevigata) could limit its ability to respond to such change.
Our findings also suggest that climatic warming could threaten the persistence of B. perstellata given its limited plasticity in response to warming (Figure 3).Although there were some observable differences in plasticity among populations of both Boechera species (Fig- ures 2 and 3), the insignificance of these differences could reflect overall similarities given their shared evolutionary history or be due to relatively small sample sizes within populations that reduced our statistical power to detect population-level differences compared with differences at the species level.
Importantly, phenotypic plasticity is not always adaptive (Bonser, 2021; Hendry, 2016;Palacio-López et al., 2015), but research on the adaptive nature of phenotypic plasticity in plant traits has been limited (Arnold et al., 2019;Wei et al., 2021), including within the context of rarity (Boyd, Anderson, et al., 2022).While our selection analyses revealed that plasticity was not always influential to fitness (i.e., plasticity was most neutral), we did find evidence that plasticity in RSR mass across Boechera species within the context of water availability is adaptative.Plasticity in root allocation as a response among plants to changes in the availability of water and other resources (Eriz et al., 2017) is a common example of the optimal partitioning theory (see Bloom et al., 1985).Although there is generally a positive association between vegetative size and reproductive output in plant individuals of the same age and species (Weiner et al., 2009), we concede that the use of total biomass as a fitness proxy in our selection analyses may not have resolved the actual fitness consequences of plasticity.Biomass allocation, in particular, involves trade-offs in investment toward functions including maintenance, growth, and reproduction (Weiner et al., 2009), and we were unable to resolve the relationship between vegetative and reproductive biomass of Boechera species in our experiments due to time and space constraints associated with the growth chambers.We suggest that future longer-term research experiments that perhaps utilize garden and/or field settings include assessments of more direct and lifetime fitness consequences of phenotypic plasticity (Anderson et al., 2021;Baythavong et al., 2011;Baythavong & Stanton, 2010;Van Buskirk & Steiner, 2009).
In contrast to our plasticity results, our analysis of the specieslevel genetic diversity of rare B. perstellata and widespread B. laevigata largely supported our hypothesis about the comparative genetic diversity of these rare and common species, which aligns with the results of previous comparative syntheses (Boyd, Anderson, et al., 2022;Cole, 2003;Gitzendanner & Soltis, 2000;Leimu & Fischer, 2008).Specifically, there were more than twice as many alleles present across sampled loci for widespread B. laevigata compared with rare B. perstellata even though fewer B. laevigata populations were sampled.However, our hypothesis that the widespread species would have higher levels of genetic diversity was not consistently supported at the population level given that the three sampled B. laevigata populations diverged greatly in genetic diversity, probably because they represent a range of reproductive modes.
Nearly every individual of the highly heterozygous B. laevigata population from Illinois shared an identical heterozygote genotype (i.e., fixed heterozygosity) for each polymorphic locus, resulting in a low number of multilocus genotypes and suggesting that this population is engaging in apomixis rather than sexual reproduction.High levels of heterozygosity tend to be associated with apomixis in Boechera species (Beck et al., 2012;Li et al., 2017;Rushworth et al., 2011Rushworth et al., , 2018) ) and are thought to be the result of hybridization between genetically distinct lineages (Alexander et al., 2015;Beck et al., 2012;Dobeš et al., 2006;Li et al., 2017;Windham et al., 2015).Although TA B L E 1 Measures of genetic diversity of individuals grown in growth chambers from seed collected from four natural populations of rare Boechera perstellata and three populations of widespread B. laevigata.(Nei, 1987) are weighted by sample size.
It has been broadly hypothesized that species that can tolerate a wide range of environmental conditions (i.e., species with broad niche breadths) would be associated with relatively wide geographic range sizes and diverse habitat types (Brown, 1984;Cardillo et al., 2019;Gaston, 2000;Slatyer et al., 2013).Previous research on rare species reported a positive association between phenotypic plasticity (within the context of edaphic factors) and niche breadth (Rutherford et al., 2017), and this association has been more broadly demonstrated with congeneric comparisons beyond the context of rarity species (e.g., Bell & Sultan, 1999;Griffith & Sultan, 2005;Sultan, 2001).While the plasticity of rare endemic B. perstellata in response to water suggests that its fundamental niche may be broader than its current distribution, which could facilitate its persistence in the face of some types of abiotic disturbance, its limited genetic

O PEN R E S E A RCH BA D G E S
This article has earned an Open Data badge for making publicly available the digitally-shareable data necessary to reproduce the reported results.The data is available at https://datadryad.org/stash/ share/AlNwOP2bbXWggvCmMrnrCl8R3X6pvYjC-eWOCNl-ViM.
Maps of the distribution of rare Boechera perstellata (a) and widespread B. laevigata (b) in the eastern United States at the county level (green shaded areas) and locations of the natural populations of each species from which seeds were collected (yellow dots).While B. perstellata only occurs in the United States, the range of B. laevigata extends into southeastern Canada.
unbiased expected heterozygosity (H e ).Examination of alleles indicated that the Illinois population (IL) of B. laevigata is likely apomictic and that the Pennsylvania population (PA) of this species experiences high levels of inbreeding.To account for possible apomixis, we used the R package Poppr v4.0.3(Kamvar et al., 2014), generating a unique multilocus genotype dataset with a clonal threshold of 0 for an analysis of clonal diversity.In addition to the number of unique multilocus genotypes (MLG), clonal diversity measures included the expected number of multilocus genotypes (eMLG), the Shannon-Wiener index of MLG diversity using natural logarithm (H;Shannon, 1948), andSimpson's (1949) index (D).To assess population structure and grouping of individuals in the study, we used a discriminant analysis of principal components (DAPC;Jombart et al., 2010;Machado et al., 2021) with the R package adegenet v2.1.3(Jombart, 2008).DAPC is a nonparametric multivariate analysis that combines principal component analysis, K-means clustering, and discriminant analysis and that does not require assumptions of populations being in Hardy-Weinberg equilibrium or the absence of linkage disequilibrium reduced soil moisture and that plasticity of root traits could facilitate the distribution of B. laevigata across a broader latitudinal range than the constrained range of B. perstellata.In contrast to their warming responses, root traits of B. perstellata and B. laevigata both responded to changes in water availability in similar directions.Their shared reductions in root biomass and allocation in response to increased watering could indicate a shift away from strategies that would enhance the acquisition of belowground resources when those resources become more plentiful, which could facilitate their association with habitats ranging from fairly dry to moist.We suggest that the stronger plasticity of root traits in response to water availability of B. laevigata could elucidate its association with a wider range of substrates than rare B. perstellata.The only trait for which rare B. perstellata exhibited more plasticity than B. laevigata was SLA in response to increased water diversity and limited plasticity in the context of temperature could suggest that it is less able to adapt and/or acclimate to the range of temperatures experienced by widespread B. laevigata across its broader range.Although the growth chamber-based approach of our research allowed for tight controls of environmental conditions across which plasticity could be assessed, future transplant experiments of narrowly distributed rare species such as B. perstellata beyond their natural ranges could be used to further assess their fundamental niche size compared with more common congeners in "real world" conditions.Such transplants combined with replacement and/or additive experiments also could elucidate the potential role of local edaphic factors and biotic factors such as competition in the restriction of B. perstellata to its narrow range and habitat type, especially given the potential for invasive non-native and prolific native species to encroach into its habitat in response to disturbance(USFWS, 2004).Although our study was focused on just one pair of congeneric species, our experimental approach combined with "real world" transplants could be applied to other rare species toward elucidating broader understanding of species rarity.Such work would need to consider the protected status and imperiled nature of many rare species and integrate careful plans for both research and protection.AUTH O R CO NTR I B UTI O N SJennifer Nagel Boyd: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (lead); writing -original draft (lead); writing -review and editing (lead).Carol Baskauf: Conceptualization (equal); data curation (equal); formal analysis (equal); TA B L E 3 Clonal diversity statistics using a clonal threshold of 0 for individuals grown in growth chambers from seed collected from four natural populations of rare Boechera perstellata and three populations of widespread B. laevigata.
funding acquisition (equal); supervision (lead); writing -original draft (supporting); writing -review and editing (supporting).Annie Lindsay: Data curation (equal); writing -review and editing (supporting).JillT.Anderson: Conceptualization (equal); formal analysis (equal); writing -original draft (supporting); writing -review and editing (supporting).Jessica Brzyski: Conceptualization (equal); funding acquisition (equal); writing -review and editing (supporting).Jennifer Cruse-Sanders: Conceptualization (equal); funding acquisition (equal); writing -original draft (supporting); writing -review and editing (supporting).ACK N OWLED G M ENTS This research was supported by the National Science Foundation Awards 1655762 and 1655521, an Austin Peay State University (APSU) Graduate Research Support Grant, and the APSU Department of Biology.The authors thank J. Odell and W. Rogers for field collections of seeds of B. perstellata and B. laevigata; E. Bach, S. Baskauf, L. Deason, K. England, E. Faulkner, C.D. Fitch, Z. Irick, B. Isaac, J. Isaac, A. Malcom, L. Malcom, E. Raskin, M. Robinson, J. Simons, T. Witsell, and J. Whittle for field collections of B. laevigata leaves and/or seeds; J. Odell for assistance with the growth chamber experiments; C.D. Finch, S. Thrasher, L. Deason, and C. Kelly for assistance with genetics work; G. Call, T. Crabtree, K. McDonald, and T. Littlefield for facilitation of site locations and access for seed collections.
to compare the genetic diversity of B. perstellata and B. laevigata in terms of allelic richness (Rs, a measure of the number of alleles independent of sample size) and sample-size weighted Across all treatments, 84.5% of B. perstellata individuals and 92% of B. laevigata individuals used in our plasticity experiment survived to harvest (Table Measures include the number of alleles per locus, number of private alleles, percentage of polymorphic loci, direct count of heterozygosity (H o ), and Nei's, 1978 unbiased estimate of mean expected heterozygosity (H e ).Values shown are means across 17 microsatellite loci ± standard errors (when applicable).Populations of B. perstellata: Franklin County, Kentucky, USA (KY1, KY2); Rutherford County, Tennessee, USA (TN1); Smith County, Tennessee (TN2).Populations of B. laevigata: Cook County, Illinois, USA (IL); Clarion County, Pennsylvania, USA (PA); Cheatham County, Tennessee, USA (TN).Statistical comparison of several genetic diversity statistics averaged across 17 microsatellite loci for four Boechera perstellata and three Boechera laevigata populations, using FSTAT.For the presumed apomictic population of B. laevigata, only unique MLGs (n = 9) were included in the analysis.R S is the expected number of alleles per locus at the smallest number of sampled individuals at a locus.H o and H s TA B L E 2 Statistics include the number of multilocus genotypes, expected number of multilocus genotypes at the least common sample size ≥10 based on rarefaction, Shannon-Wiener index of multilocus genotype diversity (H), and Simpson's index of diversity (D).Values shown are means across 17 microsatellite loci ± standard errors (when applicable).Populations of B. perstellata: Franklin County, Kentucky, USA (KY1, KY2); Rutherford County, Tennessee, USA (TN1); Smith County, Tennessee (TN2).Populations of B. laevigata: Cook County, Illinois, USA (IL); Clarion County, Pennsylvania, USA (PA); Cheatham County, Tennessee, USA (TN).